The object of this research is to increase our understanding of the mechanism of RNA polymerase. We are using various spectroscopic techniques (circular dichroism, absorption spectra, fluorescence, electron paramagnetic resonance and laser Raman spectroscopy) to study the conformation of RNA polymerase, both the core enzyme and holo enzyme, in order to assess the role of sigma factor. In addition to the intrinsic spectroscopic properties of the protein, various probes are being used-rifampicin, modified rifamycins with spin and fluorescence labels attached, and dyes such as Congo Red, aurin tricarboxylic acid and gallin. These physical techniques and probes will be used to follow the reaction of polymerase with DNA to determine the kinetics of binding and the conformational state of the enzyme and DNA as a function of temperature, ionic strength and nature of the template. Chemical modification procedures on the intact enzyme and isolated subunits as well as photocrosslinking experiments will be used to probe the mechanism of DNA binding. The kinetics and conformational changes associated with binding of nucleoside triphosphates to DNA-enzyme complexes will be studied also to gain further insight into the initiation process. Conformation properties of the chain elongation complex will also be explored.